DESCRIPTION: The pancreatic ATP-sensitive potassium channels (KATP channels) control insulin secretion in B-cells. The long-term goal is to understand how KATP channels are regulated at the molecular level to control insulin secretion. In addition to being regulated by intracellular ATP and ADP, a property, which enables the channel to couple, cell energy to cell excitability, the investigators recently showed that the channel is profoundly regulated by membrane phosphoinositides. Phosphoinositides modulate the sensitivity of KATP channels to ATP over several orders of magnitude. This modulation provides a mechanism to allow channels to be gated by physiological concentrations of ATP and ADP. Relative to regulation by intracellular nucleotides, our understanding of channel regulation by phosphoinositides is still in its infancy. The research proposed is aimed at understanding the physiological significance and the molecular basis of this novel regulatory mechanism. To assess the physiological significance, the investigators will manipulate membrane phosphoinositide levels in insulin-secreting cells and-examine the effects on channel activity and insulin secretion. The investigators will also disrupt the interaction between phosphoinositides and channels in these cells by overexpressing mutant channels lacking the ability to interact with phosphoinositides, and inhibitory peptides that compete with endogenous channels for phosphoinositide binding, and evaluate the physiological outcome. The investigators will determine the structural elements within the channel proteins involved in phosphoinositide regulation using systematic site-directed mutagenesis approach combined with electrophysiolgical measurements that monitor functional interaction between the channel and phosphoinositides, and with biochemical measurements that monitor the physical interaction between the channel and phosphoinositides. The information we propose to obtain is crucial for understanding KATP channel regulation and, hence, insulin secretion. It will provide insights to novel therapeutic approaches to diseases of insulin secretion.